Project Summary/Abstract Cellular proteins that rejoin the ends of broken chromosomes at sites called DNA double-strand breaks (DSBs) are vital because inefficient repair of such lesions leads to mutations and chromosome instability. Several human genetic disorders have been linked to defects in this type of DNA repair and have been shown to predispose affected individuals to development of cancer and/or premature aging. The goal of the proposed work is to improve our understanding of the genes and metabolic pathways required for efficient repair of broken DNA. Specific objectives are to investigate new genes identified in a genome-wide genetic search to determine their roles in the two major pathways responsible for repairing DSBs and in maintenance of DNA sequence integrity. Our unique genetic screening approach, employing two large libraries of mutant strains, has identified new genes required for repair of DSBs in the model eukaryote Saccharomyces cerevisiae (budding yeast). Each of the new mutants lacks the ability to repair DSBs induced by synthesis of a DNA strand-breaking endonuclease inside cells and after exposure to strand-breaking chemicals. These phenotypes are hallmarks of DSB repair mutants and indeed 21 known repair genes were detected in the genetic search. A total of 44 new genes were identified that have not previously been linked to DSB repair and are likely to have important functions in yeast cells and, via conserved genes with equivalent functions, in cells of higher organisms. The experiments proposed in the Aims of this proposal are designed to define the functions of these genes in the two known DSB repair pathways, which are called homologous recombination and nonhomologous end-joining (NHEJ). DNA repair and DNA mutation rates will be quantitated using genetic assays developed for those purposes. Each of the experiments has been designed to assess the functions of the new genes using relatively rapid, high throughput methods that retain the ability to yield strong statistics. The findings will spotlight those specific genes that are most critical for maintenance of genome integrity and are of greatest concern for their potential impact on DNA stability in other organisms including humans. PUBLIC HEALTH RELEVANCE: Project Narrative Many of the genes that protect DNA within human cells from mutations and rearrangements have yet to be identified. The proposed work will investigate the functions of over 40 yeast genes recently shown to protect DNA integrity. Most of the protective yeast genes share strong sequence similarity with human genes which may serve similar functions.